A system is being developed for clinical analysis using components that are significantly smaller than current analysis systems, and suitable for use in remote environments such as space flight. Current systems for assaying medically-relevant parameters of blood typically require taking one or more venous blood samples, which are then analyzed in one or more large, specialized clinical autoanalyzer systems. The systems, besides being bulky, generate significant volumes of medical waste, which must be treated as a hazardous material.
Moreover, current systems are often highly automated, and have dedicated staff to manage the flow of samples through the machine. Such systems are well adapted to hospitals and clinics. However, there are numerous situations which require more flexibility in a clinical analyzer, and some situations have a relatively low volume of sampling, requiring an analyzer which is suitable for intermittent use. Such requirements may be presented by isolated populations or locations.
Such a machine must be “robust” in operation, so that it can be used without extensive training. Moreover, it should avoid or minimize the amount of waste generated by minimizing the need for large samples and flexibly running multiple assays on a single small sample—for example, a finger prick vs. a sample from a vein. In addition, the system should have the ability to be used only intermittently, rather than daily or continuously as in most current analyzers. The system should also be flexible to allow a wide variety of assays to be analyzed.
One important part of a system for accomplishing these objectives is a device and a method to accomplish all of the steps needed for measurement of clinical parameters, including sample dilution, mixture of a sample with reagents, and delivery of the diluted sample to a flow cell for quantification of one or more parameters. Another important aspect of the system is the ability to work with small samples of blood or other bodily fluid, with sample volumes in the sub-milliliter range, for example 3 to 100 microliters. A related aspect of the system is the ability to perform continuous fluid flow at low differential pressures, to prevent leakage of components and for safety. Such a device is the subject of the present invention.
Another important requirement for the handheld diagnostic system is the need for mixing a blood sample with a stream of analytical reagents. In particular, there is a need for inline mixing in the instrument of the invention. A small sample—typically 3 to 100 microliters, for example from a finger prick—is optionally diluted and fed into an analyzer of the invention.
There are several methods described in the art for mixing microfluidic streams in a flow chamber. These are briefly discussed in Chen and Jang (“Recent Patents on Micromixing Technology”, Recent Patents on Mechanical engineering 2009, 2, 240-247).
A first alternative is a Dean effect spiral, as described for instance in Ji et al (U.S. Pat. No. 7,160,025) and Sundarsan and Ugaz (US 2007/026377A1). When streams of two fluids of similar properties are brought together non-turbulently, they mix primarily by diffusing. This is a slow process. The process can be accelerated by using “Dean Mixing” in a curved path. In a curved path, mixing occurs even in a laminar flow regime. The Dean effect is non-turbulent and so has a relatively low pressure drop. However, it still takes a significant length of flow path, with a corresponding cost in pressure drop.
A second alternative is an expansion effect in a stream, as described in Sudarsan & Ugaz PNAS 103 (19) 7228-7233; 2006). In this system, a sudden expansion of channel cross section, and if required a subsequent return to a small diameter channel, can mix fluid streams in a short path length. However, the flow tends to be turbulent, causing a relatively high pressure drop in a short distance, and requiring extended rinsing times during cleaning, because the flow is not predictable.
A third alternative is a three dimensional vortex mixer, which has one or more out-of-plane channels for generating transverse flows (Lin et al, J Micromech Microeng. 15 935-943, 2005). In an embodiment useful in the present invention, flow enters through a narrow channel into the beginning of a larger channel which is oriented at a right angle (or other non-obtuse angle) to the flow in the narrow channel. Flow initially spirals around the wall of the larger channel and gradually becomes a flow moving along the larger channel.
A fourth alternative, related to the vortex mixing system, is an out-of-plane drop, approximately equivalent to a right-angle bend. If the path diameter changes substantially, it can be equivalent to a vortex. However, if the bend is sharp and diameter change is limited, it can produce turbulent mixing with relatively low pressure drop. Like other systems having turbulence, abrupt random changes in pressure and flow rate can occur.
Many micromixers for on-chip mixing have been developed based on different mechanisms to disturb the laminar flow (Nguyen and Wu, 2005; Ottino and Wiggins, 2004). These micromixers can be divided in to two groups: passive and active micromixers (Nguyen and Wu, 2005). Active mixers rely on some form of external force to generate a chaotic flow pattern in the microchannel. Some typical external sources are pressure, temperature, electrohydrodynamics, dielectrophoresis, electrokinetics, magnetohydrodynamics and acoustics. The requirement of external power sources for active micromixers makes them less feasible for most point-of-care applications. Passive micromixers rely on geometrical layout of the microchannels to cause lamination and/or chaotic advection (Ansari et al., 2010; Chen and Meiners, 2004; Munson and Yager, 2004; Stroock et al., 2002b; Sudarsan and Ugaz, 2006a, b). Lamination-based designs split the streams and rejoin them after a certain distance, thus increase total contact area between streams (Ansari et al., 2010; Munson and Yager, 2004). The staggered-herringbone design is arguably the most visible one of chaotic advection designs (Stroock et al., 2002b). Changing of flow resistance at different directions causes the streams to rotate inside the channel. However, despite its effectiveness on mixing, the staggered-herringbone grooves can potentially trap blood cells and has been used in this manner for cancer cells (Stroock et al., 2002a). Other designs include planar spiral microchannels tested by Sudarsan and Ugaz (Sudarsan and Ugaz, 2006a, b). Secondary Dean flow occurs in spiral microchannels due to their curvature, while the sudden expansion of the channel causes vortices. Mixers with three-dimensional vortex micromixers have also been utilized to generate transverse flows for rapid mixing. One such approach mixes fluids from eight individual channels tangent to a three-dimensional circular chamber (Lin et al., 2005). Other approaches have demonstrated different number of tangential inlets, from one (Long et al., 2009) to as many as sixteen (Bohm et al., 2001).
We have found that it is advantageous to combine more than one type of mixer into a sample mixing path, particularly for use in a device such as a hand-held diagnostic machine, in which weight and volume are both constrained. When properly designed and proportioned, a path can be constructed that mixes two streams (sample and analytical reagents) in a short distance at low pressure drop.
A related feature of the devices of the invention is an inlet port that minimizes differences in sample dilution, and the effect of variances in dilution, when samples of varying volume are fed into the system. A significant portion of the variance in dilution can be achieved by proper construction of the inlet into the system, so that approximately identical volumes of sample and reagents are mixed on entry.